Whole-protein screening and multi-modal profiling of antigen-specific CD4⁺ T cells at single-cell resolution

Systematic whole-protein screening and comprehensive profiling of antigen-specific CD4⁺ T cells are crucial for advancing vaccine design and cancer immunotherapies, yet remain technically challenging. Here, we present a high-throughput platform that utilizes large-scale class II single-chain trimer libraries to detect antigen-specific CD4⁺ T cells, while simultaneously profiling their antigen specificity, TCRα/β sequences, MHC restriction, whole transcriptomes, and patient/timepoint origins at single-cell resolution. Upon rigorous platform validation, we screened the full SARS-CoV-2 spike receptor binding domain in a longitudinal cohort of 22 participants, identifying 2,188 antigen-specific CD4⁺ T cells and showing key metrics defining the immunogenicity of class II-restricted viral antigens. We further extended the platform to whole-protein screening of HPV-16 E6/E7 in a cohort of precancerous patients, indicating HPV-specific CD4 TCRs that, upon extensive characterization, demonstrate strong therapeutic potential. By integrating high-throughput antigen screening with high-dimensional, multi-modal cellular characterization, our approach provides detailed insight into CD4⁺ T cell immunity, potentially guiding vaccine design and next-generation TCR-based cancer immunotherapies.